Our laboratory is studying the 70-kDa class of heat shock proteins (Hsp70s and Hsc70s) which act as molecular chaperones, that is, are involved in the ATP-dependent folding and unfolding of proteins, the formation and dissolution of protein complexes, and the translocation of proteins across membranes. In many of these processes members of the DnaJ class of proteins act as cofactors to the Hsc70 proteins, apparently "presenting" protein substrates to Hsc70. One of the processes where a DnaJ homolog is required for Hsc70 action is the uncoating of clathrin-coated vesicles. In studying the mechanism of action of Hsc70 in uncoating bovine brain clathrin-coated vesicles, we discovered that the protein cofactor, auxilin, is required for the uncoating process to occur, and we also showed that auxilin is a DnaJ homolog. In comparing the direct interaction of auxilin and other DnaJ homologs with Hsc70, we found that, as we showed with other DnaJ homologs, auxilin increases the rate of the ATP hydrolysis step and causes reversible polymerization of Hsc70 in the presence of ATP. This latter effect may represent presentation of one Hsc70 to another by DnaJ homologs under conditions where substrates are not present. However, we also found that, while most DnaJ homologs bind quite weakly to Hsc70 in ATP, auxilin binds much more strongly. We are currently investigating what portion of auxilin is responsible for this strong binding to Hsc70. We are also investigating what portion of auxilin and other DnaJ homologs prevents them from interacting with Hsc70 in ADP. In our studies on the uncoating reaction, we have also found that, following one round of uncoating, some type of complex of auxilin, assembly proteins, and soluble clathrin triskelions forms which strongly inhibits further uncoating by Hsc70. We are currently investigating the nature of this complex which may be important physiologically as a nucleating complex that does not polymerize in the cytosol in the presence of Hsc70 but facilitates formation of coated pits in nerve cells which carry out rapid endocytosis. Finally, we have found that overexpression of auxilin in either Cos cells or HeLa cells inhibits endocytosis by these cells. This effect does not depend on the interaction of Hsc70 with auxilin because it also occurs with auxilin mutated in the DnaJ region so that it no longer interacts with Hsc70 in vitro. In addition, an even stronger inhibition of endocytosis occurs with the brain-specific assembly protein, AP-180. In contrast to these results, it has been reported that the addition of the assembly protein, AP-2, to permeabilized cells, increases the rate of endocytosis. Therefore, our data suggest that overexpression of brain-specific assembly proteins in cells which normally do not express these proteins may inhibit endocytosis.